Abstract of the PDB Structure's related Publication:
N 6 -methyladenosine (m 6 A) is an abundant modification in messenger RNA and noncoding RNAs that affects RNA metabolism. Methyltransferase-like protein 16 (METTL16) is a recently confirmed m 6 A RNA methyltransferase that methylates U6 spliceosomal RNA and interacts with the 3'-terminal RNA triple helix of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). Here, we present two X-ray crystal structures of the N-terminal methyltransferase domain (residues 1-291) of human METTL16 (METTL16_291): an apo structure at 1.9 Å resolution and a post-catalytic S-adenosylhomocysteine-bound complex at 2.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class I S-adenosylmethionine-dependent methyltransferases and a large, positively charged groove. This groove likely represents the RNA-binding site and it includes structural elements unique to METTL16. In-depth analysis of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. In contrast to the major m 6 A methyltransferase heterodimer METTL3/METTL14, full-length METTL16 forms a homodimer and METTL16_291 exists as a monomer based on size-exclusion chromatography. A native gel-shift assay shows that METTL16 binds to the MALAT1 RNA triple helix, but monomeric METTL16_291 does not. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.
METTL16 is an RNA N6-methyltransferase that selectively modifies adenosine residues at their N6 position in a discrete group of RNAs, contributing to the regulatory mechanism governing S-adenosyl-L-methionine homeostasis through the modulation of MAT2A transcript levels ( Pendleton et al. 2017, Mendel et al. 2018, Doxtader et al. 2018, Yu et al. 2021, Mendel et al. 2021). It is proficient in N6-methylating a subset of messenger RNAs (mRNAs) as well as U6 small nuclear RNAs (U6 snRNAs) ( Pendleton et al. 2017). Unlike the METTL3-METTL14 heterodimer, METTL16 exhibits specificity, requiring both a 5'UACAGAGAA-3' nonamer sequence and a particular RNA conformation for methylation ( Pendleton et al. 2017, Mendel et al. 2018, Doxtader et al. 2018). It plays an instrumental role in the homeostasis of S-adenosyl-L-methionine. In conditions of S-adenosyl-L-methionine abundance, METTL16 interacts with the 3'-UTR of MAT2A mRNA and specifically N6-methylates the first hairpin, thereby preventing U2AF1/U2AF35 from recognizing the 3'-splice site and subsequently inhibiting the splicing and protein production of S-adenosylmethionine synthase ( Pendleton et al. 2017, Mendel et al. 2021). Under S-adenosyl-L-methionine-limited conditions, it still binds to the 3'-UTR of MAT2A mRNA but stalls due to the absence of a methyl donor, thereby promoting MAT2A expression ( Pendleton et al. 2017 ). Beyond mRNAs, it also mediates the N6-methylation of U6 snRNA, specifically targeting adenine at position 43 ( Pendleton et al. 2017 , Warda et al. 2017, Aoyama et al. 2020).